Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion-gradients, osmotic balance, action potentials and apoptosis. The proposed work will address key questions regarding the mechanisms of nutrient uptake in Escherichia coli and other Gram negative bacteria. In E. coli, rare nutrients are sequestered by specific outer- membrane proteins that derive energy by coupling to the inner-membrane protein TonB. These TonB-dependent transporters include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. TonB-dependent transporters are abundant in Gram negative bacteria and are critical to the success of many bacterial pathogens, such as the bacteria that result in meningitis, cholera, pertussis and dysentery. Because they are unique to bacteria, these transporters are a rational target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters; however, the mechanism by which transport takes place is unclear. The proposed work will determine the mechanisms by which protein-protein interactions are regulated in this system and test models for the molecular mechanisms of transport. Site-directed spin labeling and EPR spectroscopy in combination with high-resolution NMR will be used to examine ligand- induced structural changes, dynamics and conformational exchange within these transporters. The proposal will use novel approaches test for conformational dynamics and map changes in the energy landscape within these membrane proteins. Approaches will be used to increase the population of excited conformational states so that intermediate structures in the transport process may be characterized.